The present invention relates to a method of expanding and maintaining embryonic stem cells (ESCs) in an undifferentiated state in a suspension culture, and more particularly, to methods of using such ESCs for the generation of lineage-specific cells which can be used for cell-based therapy.
Human embryonic stem cells (hESCs) are proliferative, undifferentiated stem cells capable of differentiating into cells of all three embryonic germ layers. As such, hESCs hold promise for various applications including cell-based therapy, pharmaceutical screening, identification of drug targets and cell-based compound delivery which require almost indefinite amounts of proliferating, yet pluripotent hESCs.
To facilitate the exploitation of hESCs in both cell-based therapy and use in the pharmaceutical industry for drug screening, identification of drug targets and cell-based compound delivery, hESCs cultures should be scaled-up and optimized. However, culturing of hESCs on any of the currently available 2-dimensional (2-D) culturing systems (i.e., feeder layers or feeder-free matrices) limits the expansion capacity of the cells. On the other hand, when ESCs are removed from their feeder layers or feeder-free matrices and transferred to common suspension cultures, the cells loose their undifferentiated state and rapidly differentiate (Thomson et al., 1998). Thus, culturing of hESCs in suspension in Petri dishes usually results in the formation of aggregates containing differentiating cells termed embryoid bodies (EBs) [Itskovitz-Eldor et al, 2000].
To overcome such limitations, Fok and Zandstra (Fok E Y, and Zandstra P W, Stem Cells. 2005, 23: 1333-42) developed stirred-suspension cultures in which the ESCs are attached to glass microcarriers. However, although ESCs cultured under such conditions exhibited typical ESC expression patterns and retained the developmental potential of the starting cell population, the technical difficulties associated with adherence and dissociation of the ESCs from the microcarrier surface limit the robustness potential of such a culturing method. Another study by Gerecht-Nir and Itskovitz-Eldor (disclosed in PCT/IL03/01017) describes a dynamic culturing system for differentiating embryoid bodies or expanding ESCs under non-differentiation conditions. In this system, ESCs are seeded in a bioreactor designed to exert random gravity forces. However, PCT/IL03/01017 does not teach non-dynamic suspension culture systems. Another study by Cormier J. et al. (Tissue engineering 12: 3233-3245, 2006) describes culturing for 6 days of mouse embryonic stem cells (mESCs) in a suspension culture in the presence of leukemia inhibitory factor (LIF) and bovine serum under constant agitation. In a later publication (Zur Nieden N I, et al., 2007; J. of Biotechnology 129: 421-432) the authors reported that mESCs cultured in suspension under static conditions and using trypsin for passaging every 2 days exhibited a sharp decrease in the expression of undifferentiated markers such as Oct-4 and failed to maintain pluripotency as detected by the expression of early ectodermal and endodermal differentiation markers. In addition, the doubling time of the mESCs that were cultured in the dynamic or static suspension cultures was only 15 hours (Zur Nieden., et al., Supra), which may lead to chromosomal instability and abnormality (Cowan C A., et al., 2004, N. Engl. J. Med. 350: 1353-1356). In addition, in contrast to mESCs, it is known that LIF cannot maintain human ESCs in an undifferentiated state (Thomson et al, 1998; Reubinof et al, 2000). Thus, to date, continuous culturing of undifferentiated human ESCs in suspension under conditions devoid of substrate adherence (e.g., a carrier) was never demonstrated.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of obtaining a scalable culture of hESCs devoid of the above limitations.